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1.
Anesthesiology ; 95(4): 857-61, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11605924

ABSTRACT

BACKGROUND: Ambulatory epidural analgesia (AEA) is a popular choice for labor analgesia because ambulation reportedly increases maternal comfort, increases the intensity of uterine contractions, avoids inferior vena cava compression, facilitates fetal head descent, and relaxes the pelvic musculature, all of which can shorten labor. However, the preponderance of evidence suggests that ambulation during labor is not associated with these benefits. The purpose of this study is to determine whether ambulation with AEA decreases labor duration from the time of epidural insertion to complete cervical dilatation. METHODS: In this prospective, randomized study, 160 nulliparous women with AFA were randomly assigned to one of two groups: AEA with ambulation and AEA without ambulation. AEA blocks were initiated with 15-20 ml ropivacaine (0.07%) plus 100 microg fentanyl, followed by a continuous infusion of 0.07% ropivacaine plus 2 microg/ml fentanyl at 15-20 ml/h. Maternal measured variables included ambulation time, time from epidural insertion to complete dilatation, stage II duration, pain Visual Analogue Scale scores, and mode of delivery. APGAR scores were recorded at 1 and 5 min. Results are expressed as mean +/- SD or median and analyzed using the t test, chi-square, or the Mann-Whitney test at P < or = 0.05. RESULTS: The ambulatory group walked 25.0 +/- 23.3 min, sat upright 40.3 +/- 29.7 min, or both. Time from epidural insertion to complete dilatation was 240.9 +/- 146.1 min in the ambulatory group and 211.9 +/- 133.9 min in the nonambulatory group (P = 0.206). CONCLUSION: Ambulatory epidural analgesia with walking or sitting does not shorten labor duration from the time of epidural insertion to complete cervical dilatation.


Subject(s)
Analgesia, Epidural , Analgesia, Obstetrical , Early Ambulation , Labor, Obstetric/physiology , Adult , Apgar Score , Female , Humans , Infant, Newborn , Pain Measurement , Pregnancy , Pregnancy Outcome , Prospective Studies , Time Factors
2.
Anesthesiology ; 95(3): 585-93, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11575528

ABSTRACT

BACKGROUND: Abundant in vitro and animal model data suggest the postsynaptic gamma-aminobutyric acid receptor type A (GABA(A)-R) is an important target for volatile general anesthetics, but the relevance of these models is untested in humans. Because benzodiazepines have also been shown to act via a specific GABA(A)-R site, they provide sensitive probes for the GABA(A)-R. Availability of the 11C-labeled benzodiazepine ligand, flumazenil, allowed us to quantitatively test in humans whether the volatile anesthetic isoflurane affects GABA(A)-Rs in vivo in a dose-dependent manner. METHODS: 11C-flumazenil positron emission tomography scans were obtained in 12 healthy subjects while awake (control condition) and anesthetized with either 1.0 or 1.5 minimum alveolar concentration isoflurane (n = 7 and 5, respectively; isoflurane conditions). Regions of interest included areas of high, intermediate, and low GABA(A)-benzodiazepine site density. For each subject and experimental condition, the binding of 11C-flumazenil, expressed as distribution volume (which linearly correlates to maximal binding site density and apparent ligand affinity), was obtained by curve fitting using a two-compartment model. RESULTS: The ratio of distribution volume increased significantly in each examined region during the isoflurane conditions compared with control conditions (P < 0.01, one-tailed t test). Furthermore, the increases in ratio of distribution volume during the 1.5-minimum alveolar concentration isoflurane condition were significantly greater than those measured during 1.0 minimum alveolar concentration isoflurane inhalation (P < 0.002, one-tailed t test). CONCLUSIONS: Isoflurane exposure appeared to enhance receptor-specific 11C-flumazenil binding in a dose-dependent manner. The results suggest the possibility that a conformational change of the GABA(A)-R is involved in the mechanism of action of isoflurane in the living human brain.


Subject(s)
Anesthetics, Inhalation/pharmacology , Isoflurane/pharmacology , Receptors, GABA-A/drug effects , Adult , Blood-Brain Barrier , Cerebrovascular Circulation/drug effects , Chlorides/metabolism , Dose-Response Relationship, Drug , Female , Flumazenil/metabolism , Humans , Male , Protein Binding , Receptors, GABA-A/metabolism
3.
Biochim Biophys Acta ; 1509(1-2): 111-22, 2000 Dec 20.
Article in English | MEDLINE | ID: mdl-11118523

ABSTRACT

The trend of evidence suggests that general anesthetics act directly on proteins in the neural membrane. However, the fact that the functions of nicotinic acetylcholine receptor (sodium permeability, desensitization rate) are modulated by the composition of the membrane in which it is reconstituted has been thought to be a result of the variation of interactions between acetylcholine receptor and membrane. In this study, protein-lipid interaction at the level of the lipid headgroup was investigated using electron paramagnetic resonance (EPR) and headgroup spin label. Lipid headgroup mobility was evaluated with rotational correlation time from the EPR spectrum. Protein-lipid interaction at headgroup depth was demonstrated from the motionally restricted component of the spectrum. Rotational correlation time increased to 13 ns from 7 ns due to protein-lipid interaction. The effect of anesthetic (ethanol, 1-hexanol, and isoflurane) on protein-lipid interaction was investigated, and the correlation time was 13 ns. It is concluded that the anesthetics used in this study did not alter protein-lipid interaction at the level of the lipid headgroup, so far as observed by rotational correlation time, without excluding the possibility that anesthetics that perturb protein-lipid interactions modulate receptor functions via this mechanism.


Subject(s)
Alcohols/pharmacology , Isoflurane/pharmacology , Lipid Bilayers/chemistry , Lipids/chemistry , Receptors, Nicotinic/chemistry , Anesthetics, Inhalation/pharmacology , Electron Spin Resonance Spectroscopy , Ethanol/pharmacology , Hexanols/pharmacology , Phosphatidylcholines/chemistry , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/isolation & purification , Spin Labels
4.
Br J Anaesth ; 85(5): 757-62, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11094594

ABSTRACT

On the basis of data implicating GABAA receptors in the effects of volatile general anaesthetics, we hypothesized that alcohol-, barbiturate-, and benzodiazepine-sensitive alcohol-nontolerant (ANT) rats would also be more sensitive than alcohol-tolerant (AT) rats to two clinical general anaesthetics with differing potencies, halothane and desflurane. The obtunding effect of halothane and desflurane on mature ANT (n = 17) and AT (n = 16) rats was assessed by the loss-of-righting reflex endpoint. ANT rats were significantly (P < 0.0001) more sensitive to the obtunding effects of both halothane and desflurane (ED50 = 0.45 +/- 0.03% atm for ANT vs 0.95 +/- 0.04% atm for AT and 2.16 +/- 0.17 vs 3.69 +/- 0.13% atm, respectively). The immobilization effect of halothane and desflurane was assessed with the tail clamp/withdrawal endpoint. ANT rats were more sensitive to the effects of halothane (ED50 = 1.10 +/- 0.08% atm for ANT vs 1.72 +/- 0.09% atm for AT; P < 0.0001) but not desflurane (ED50 = 6.25 +/- 0.25% atm for ANT vs 5.85 +/- 0.21% atm for AT). The data presented support the hypothesis that volatile anaesthetics interact with specific neuronal proteins (possibly GABAA receptors) and agree with recent hypotheses that different elements of the anaesthetic state are produced by separate sites or mechanisms.


Subject(s)
Anesthetics, Inhalation/pharmacology , Halothane/pharmacology , Isoflurane/analogs & derivatives , Receptors, GABA-A/drug effects , Animals , Desflurane , Dose-Response Relationship, Drug , Drug Tolerance/genetics , Ethanol/pharmacology , Isoflurane/pharmacology , Male , Movement/drug effects , Rats , Rats, Long-Evans , Rats, Mutant Strains , Rats, Sprague-Dawley , Rats, Wistar , Receptors, GABA-A/physiology , Reflex/drug effects
5.
Pharmacol Biochem Behav ; 66(2): 371-4, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10880692

ABSTRACT

The gamma 2 subunit is required for benzodiazepine modulation of the GABA(A) receptor. Alternate splicing of precursor GABA(A) gamma 2 mRNA results in two splice variants, a short (gamma 2S) and a long (gamma 2L) variant. We investigated the roles of these splice variants in benzodiazepine pharmacology using mice lacking genes for the gamma 2L splice variant. Sleep time responses to midazolam and zolpidem were 20 and 18% greater, respectively, in null allele mice compared with wild-type mice, while responses to nonbenzodiazepine agents such as etomidate and pentobarbital were unchanged. Although the GABA(A) receptor number was not altered in null allele mice, there was a corresponding increase in affinity of brain membranes for benzodiazepine agonists (midazolam, diazepam, and zolpidem), while affinity for benzodiazepine inverse agonists (beta CCM and Ro15-4513) was decreased. These changes were not observed in inbred mice of the parental strains (C57BL/6J and 129/SvJ) used to create the genetically altered mice, indicating that differences between gamma 2L null allele and wild-type mice were unlikely to be simply due to cosegregation of linked alleles. Absence of the gamma 2L splice variant increases the affinity of receptors for benzodiazepine agonists, and is associated with a modest increase in behavioral sensitivity to benzodiazepine agonists. Lack of the gamma 2L subunits may shift the GABA(A) receptor from an inverse agonist-preferring toward an agonist-preferring configuration.


Subject(s)
Benzodiazepines/pharmacology , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Alternative Splicing , Animals , Azides/pharmacology , Brain/drug effects , Brain/metabolism , Carbolines/pharmacology , Diazepam/pharmacology , Drug Resistance , Female , Flumazenil/metabolism , GABA Modulators/pharmacology , Genetic Variation , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Midazolam/pharmacology , Pyridines/pharmacology , Receptors, GABA-A/drug effects , Sleep/drug effects , Zolpidem
6.
Neuroscience ; 95(3): 795-806, 2000.
Article in English | MEDLINE | ID: mdl-10670447

ABSTRACT

A line of mice was recently created in which the gabrb3 gene, which encodes the beta3 subunit of the GABA(A) receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA(A) receptors in the CNS enabled an investigation of the role of GABA and GABA(A) receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA(A) and GABA(B) receptor agonists. Homozygous null (beta3-/-) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (beta3+/+) and heterozygous (beta3+/-) littermates. The beta3-/- mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their beta3+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in beta3-/- mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA(A) receptors in the spinal cord. As expected, subcutaneous administration of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in beta3-/- mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, beta3+/+ and beta3+/- mice. However, the antinociceptive effect of the GABA(B) receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in beta3-/- mice compared to the progenitor strains, beta3+/+ or beta3+/- mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA(A) receptors can affect the production of antinociception by other analgesic drugs as well.


Subject(s)
GABA Agonists/pharmacology , Nociceptors/drug effects , Protein Isoforms/deficiency , Receptors, GABA-A/deficiency , Sensory Thresholds/physiology , Animals , Baclofen/administration & dosage , Baclofen/pharmacology , GABA Agonists/administration & dosage , Hot Temperature , Injections, Spinal , Injections, Subcutaneous , Isonicotinic Acids/administration & dosage , Isonicotinic Acids/pharmacology , Isoxazoles/administration & dosage , Isoxazoles/pharmacology , Mice , Mice, Inbred C57BL/genetics , Mice, Inbred Strains/genetics , Physical Stimulation , Protein Isoforms/genetics , Receptors, GABA-A/genetics , Sex Characteristics
7.
Proc Natl Acad Sci U S A ; 96(22): 12905-10, 1999 Oct 26.
Article in English | MEDLINE | ID: mdl-10536021

ABSTRACT

gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.


Subject(s)
Behavior, Animal/drug effects , Hypnotics and Sedatives/pharmacology , Receptors, GABA-A/genetics , Steroids/pharmacology , Animals , Azides/metabolism , Azides/pharmacology , Benzodiazepines/metabolism , Benzodiazepines/pharmacology , Brain/drug effects , Brain/metabolism , Female , Hypnotics and Sedatives/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscimol/metabolism , Muscimol/pharmacology , Receptors, GABA-A/metabolism , Receptors, GABA-A/physiology , Sleep Wake Disorders/genetics , Steroids/metabolism
8.
Psychopharmacology (Berl) ; 145(2): 175-80, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10463318

ABSTRACT

RATIONALE: Previous fluorescence studies employing 1,6-diphenyl-1,3,5-hexatriene (DPH) have revealed an increase in the fluidity of platelet membranes from individuals with Alzheimer's disease (AD) and their first-degree relatives. This biophysical alteration has been reported to be relatively specific for the hydrocarbon core of platelet membranes, where DPH preferentially localizes; this effect is not reflected by the fluorescent reporter triethylamino-DPH, which labels membranes at the lipid-aqueous interface. OBJECTIVE: The goal of this study was to explore the validity and reproducibility of these findings using an independent biophysical technique, electron spin resonance (ESR) spectroscopy. METHODS: Platelet membranes prepared from first-degree relatives of patients with AD were labeled with DPH, or the spin-labeled fatty acid probes 5-doxylstearate (5-DS) and 12-doxylstearate (12-DS). These spin labeled probes provide an index of structural order at the respective depths of their nitroxide moieties in the membrane. The resulting preparations were examined by fluorescence and ESR spectroscopy. RESULTS: Increased platelet membrane fluidity (PMF), as determined by the fluorescence anisotropy of DPH, was associated with only a modest reduction in the order parameter derived for 5-DS labeled membranes. In contrast, the mean order parameters derived from the paired samples labeled with 12-DS differed substantially from each other, and revealed decreased order (increased fluidity) in the hydrocarbon 12-C region where DPH preferentially localizes. CONCLUSIONS: These results provide an independent validation of the biophysical alterations of platelet membranes that are manifested by a subgroup of patients with AD and their first-degree relatives.


Subject(s)
Alzheimer Disease/etiology , Blood Platelets/chemistry , Membrane Fluidity , Adult , Aged , Anisotropy , Electron Spin Resonance Spectroscopy , Female , Humans , Male , Middle Aged , Risk , Spectrometry, Fluorescence
9.
Pharmacol Biochem Behav ; 63(1): 21-6, 1999 May.
Article in English | MEDLINE | ID: mdl-10340519

ABSTRACT

Gene-targeting technology is creating an explosion in the number of animals available with single gene mutations that affect the function of the central nervous system. Most gene-targeted mice are produced on a mixed genetic background of C57BL/6J and substrains of Strain 129. Understanding the behavioral characteristics and responses to various drugs of these parental strains is vital to interpreting data from gene-targeted mice. We directly compared C57BL/6J and Strain 129/SvJ mouse lines on several behavioral paradigms and in response to several hypnotic and anesthetic drugs. Compared to Strain 129/SvJ mice, C57BL/6J animals are more sensitive to the hypnotic effects of midazolam, zolpidem, and propofol, less sensitive to etomidate and ethanol, and do not differ in sensitivity to Ro15-4513 or pentobarbital. These strains do not differ in their sensitivity to the motor ataxic effects of the volatile anesthetics enflurane or halothane. However, Strain 129/SvJs are more sensitive to the immobilizing effects of halothane but not enflurane. Motor coordination differs initially, but with repeated testing strain differences are no longer apparent. Strain 129/SvJ mice are more anxious on the elevated plus maze and open-field activity assays. Thus, these mouse strains harbor polymorphisms that influence some, but not all, traits of interest to behavioral neuroscientists.


Subject(s)
Exploratory Behavior/drug effects , Hypnotics and Sedatives/pharmacology , Maze Learning/drug effects , Psychomotor Performance/drug effects , Anesthetics/pharmacology , Animals , Ethanol/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Sleep/drug effects
10.
Neuropharmacology ; 38(2): 253-65, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10218866

ABSTRACT

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.


Subject(s)
Alternative Splicing , Brain/metabolism , Ethanol/pharmacology , Ganglia, Spinal/physiology , Genetic Variation , Neurons/physiology , Receptors, GABA-A/physiology , Animals , Anxiety , Cell Membrane/metabolism , Chimera , Crosses, Genetic , Exons , Female , Flunitrazepam/pharmacokinetics , Macromolecular Substances , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/drug effects , Neurons/drug effects , Radioligand Assay , Receptors, GABA-A/deficiency , Receptors, GABA-A/genetics , Sequence Deletion , Sleep/drug effects , Substance Withdrawal Syndrome , Transcription, Genetic
11.
Front Biosci ; 3: D548-58, 1998 Jun 08.
Article in English | MEDLINE | ID: mdl-9616129

ABSTRACT

Genetically engineered animals (e.g., transgenics, gene knockouts, gene knockins) are being utilized with increasing frequency to investigate the mechanisms of action of alcohol and anesthetics. By creating and analyzing animals that harbor precise, preplanned changes in candidate genes, researchers are rapidly making progress toward uncovering how these drugs exert their effects on the central nervous system to bring about their behavioral effects. Since these sedative / hypnotic agents are likely to exert their effects by altering neurotransmission, the majority of investigations to date have focused on neurotransmitter receptors and modulators of neurotransmission such as kinases.


Subject(s)
Anesthetics/pharmacology , Ethanol/pharmacology , Genetic Engineering , Synaptic Transmission/physiology , Alcoholic Intoxication/genetics , Alcoholic Intoxication/physiopathology , Animals , Mice , Mice, Knockout , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/physiology , Nitric Oxide Synthase Type I , Phosphotransferases/genetics , Phosphotransferases/physiology , Rats , Receptors, Adrenergic, alpha/genetics , Receptors, Adrenergic, alpha/physiology , Receptors, Dopamine/genetics , Receptors, Dopamine/physiology , Receptors, GABA-A/genetics , Receptors, GABA-A/physiology , Receptors, Neurotransmitter/genetics , Receptors, Neurotransmitter/physiology , Receptors, Opioid/genetics , Receptors, Opioid/physiology , Receptors, Serotonin/genetics , Receptors, Serotonin/physiology , Synaptic Transmission/drug effects , Synaptic Transmission/genetics
12.
Anesthesiology ; 88(3): 775-80, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9523823

ABSTRACT

BACKGROUND: The mammalian gamma-aminobutyric acid type A (GABA(A)) receptor, a likely target of anesthetic action, exhibits remarkable subunit heterogeneity. In vitro expression studies suggest that there is subunit specificity to anesthetic responses at the GABA(A) receptor. The authors tested whether genetically engineered mice that lack the beta3 subunit of the GABA(A) receptor differed in their sensitivities to several general anesthetic agents. METHODS: Median effective concentrations for loss-of-righting reflex and tail clamp/withdrawal for enflurane and halothane were determined in mice with and without the beta3 gene and gene product. Sleep time was measured after intraperitoneal injection of pentobarbital, ethanol, etomidate, and midazolam. RESULTS: Null allele mice (beta3 -/-) did not differ from wild-type mice (beta3 +/+) in the obtunding response to enflurane and halothane but were significantly more resistant to enflurane (null allele half-effect concentrations [EC50] of 2.59 +/- 0.10 vs. wild-type EC50 of 2.06 +/- 0.12 atm %, P < 0.001) and halothane (null allele EC50 of 1.73 +/- 0.04 vs. wild-type EC50 of 1.59 +/- 0.05 atm %, P = 0.01) as determined by tail clamp response. Wild-type and null allele mice exhibited divergent responses to other sedative agents active at the GABA(A) receptor. No differences were noted in sleep times after administration of pentobarbital and ethanol, but null allele mice were more resistant to etomidate (null allele EC50 of 17.8 +/- 1.9 min vs. wild-type EC50 of 26.2 +/- 2.4 min, P < 0.02) and midazolam (null allele EC50 of 14.2 +/- 7.8 min vs. wild-type EC50 of 41.3 +/- 10.4 min, P < 0.05). CONCLUSIONS: The beta3 subunit of the GABA(A) receptor appears to be important in the mediation of the immobilizing (tail clamp) but not obtunding (loss-of-righting reflex) effects of the volatile anesthetic agents enflurane and halothane. These data support the hypotheses that separate components of the anesthetic state are mediated via different central nervous system loci; that the GABA(A) receptor is a likely target for the immobilizing response to volatile anesthetic agents; and that the beta3 subunit plays a direct or indirect role in the mediation of this response. Absence of the beta3 subunit appears to attenuate the obtunding effect of midazolam and etomidate but appears not to alter the obtunding effect of pentobarbital, enflurane, and halothane, suggesting that these anesthetic agents produce hypnosis by different specific molecular mechanisms.


Subject(s)
Anesthetics, General/pharmacology , Receptors, GABA-A/deficiency , Animals , Dose-Response Relationship, Drug , Enflurane/pharmacology , Halothane/pharmacology , Hypnotics and Sedatives/pharmacology , Mice , Mice, Knockout , Receptors, GABA-A/chemistry , Reflex/drug effects , Structure-Activity Relationship
13.
Neurosci Lett ; 240(2): 81-4, 1998 Jan 09.
Article in English | MEDLINE | ID: mdl-9486477

ABSTRACT

Mice whose gamma-aminobutyric acid type A (GABA(A)) beta3 subunit gene is inactivated ('beta3 knockout mice') have been previously shown to have epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal mortality. In this study, we analyze whole-cell responses to GABA in neurons from beta3+/+, beta3+/- and beta3-/- mice. We demonstrate markedly decreased responses to GABA in both hippocampal and dorsal root ganglion neurons isolated from beta3-/- mice without major differences in the GABA concentration-response curves. We also utilize the subunit selective pharmacology of Zn2+ and the anticonvulsant drug loreclezole to help infer the presence of beta2 and gamma subunits in the GABA(A) receptors remaining in neurons from beta3-/- mice.


Subject(s)
Mice, Knockout/physiology , Neurons/metabolism , Receptors, GABA-A/deficiency , Receptors, GABA-A/genetics , Animals , Cells, Cultured , Drug Synergism , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiology , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiology , Mice , Neurons/cytology , Neurons/physiology , Patch-Clamp Techniques , Receptors, GABA-A/drug effects
14.
Can J Anaesth ; 45(2): 139-43, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9512848

ABSTRACT

PURPOSE: To measure the haemodynamic effects of rocuronium in adults undergoing cardiac surgery with cardiopulmonary bypass (CPB). METHODS: Twenty patients undergoing elective cardiac surgical procedures with moderate hypothermic nonpulsatile bypass participated in this prospective, observational study. After anaesthetic induction, recovery from succinylcholine, and achievement of baseline haemodynamic stability, patients received 0.6 mg.kg-1 rocuronium as an initial rapid intravenous bolus. Maintenance dosing of 0.2 mg.kg-1 was continued for the remainder of the procedure. Haemodynamic measurements (heart rate, systemic arterial systolic, diastolic, and mean arterial pressure, pulmonary arterial systolic, diastolic, and mean pressure, pulmonary capillary wedge pressure, central venous pressure, and thermodilution cardiac output measurements) were obtained for the first five minutes after rocuronium administration, and subjects were observed for histamine-related symptoms. RESULTS: Central venous pressure decreased from baseline at two and five minutes after the rocuronium bolus, and mean pulmonary artery pressure decreased at five minutes. No changes were observed in heart rate, mean systemic arterial pressure, pulmonary capillary wedge pressure, cardiac index, stroke volume, systemic vascular resistance, or pulmonary vascular resistance, nor did any patient manifest any other histamine-related symptoms. CONCLUSION: The haemodynamic profile for a 0.6 mg.kg-1 bolus of rocuronium is acceptable for patients with cardiovascular disease.


Subject(s)
Androstanols/pharmacology , Anesthesia, General , Cardiopulmonary Bypass , Hemodynamics/drug effects , Neuromuscular Nondepolarizing Agents/pharmacology , Adult , Aged , Aged, 80 and over , Blood Pressure/drug effects , Cardiac Output/drug effects , Female , Heart Rate/drug effects , Humans , Intraoperative Period , Male , Middle Aged , Pulmonary Artery/drug effects , Pulmonary Artery/physiology , Pulmonary Circulation/drug effects , Rocuronium
15.
Toxicol Lett ; 100-101: 301-7, 1998 Nov 23.
Article in English | MEDLINE | ID: mdl-10049157

ABSTRACT

Techniques have recently been developed that enable the creation of mice that harbor specific, predetermined genetic changes. These 'gene knockout mice', which contain a single genetic modification that is determined by the investigator, can subsequently be analyzed with tests that span the molecular, cellular, and behavioral levels. Application of such a multi-level approach to mechanisms of drug action should ultimately allow general anesthetic responses to be properly attributed to a molecular site.


Subject(s)
Anesthetics/pharmacology , Mice, Knockout/genetics , Receptors, Drug/genetics , Animals , Gene Targeting , Humans , Mice , Receptors, Drug/drug effects
16.
Mol Pharmacol ; 52(3): 380-8, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9281599

ABSTRACT

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.


Subject(s)
Cerebellum/ultrastructure , Receptors, GABA-A/classification , Receptors, GABA-A/drug effects , Animals , Azides/metabolism , Azides/pharmacology , Benzodiazepines/metabolism , Benzodiazepines/pharmacology , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carbolines/metabolism , Carbolines/pharmacology , Cerebellum/drug effects , Cerebellum/metabolism , Convulsants/metabolism , Convulsants/pharmacology , Diazepam/metabolism , Diazepam/pharmacology , Diuretics/metabolism , Diuretics/pharmacology , Furosemide/metabolism , Furosemide/pharmacology , GABA Agonists/metabolism , GABA Agonists/pharmacology , GABA Antagonists/metabolism , GABA Antagonists/pharmacology , GABA Modulators/metabolism , GABA Modulators/pharmacology , Mice , Mice, Inbred C57BL , Mutation , Pregnanolone/metabolism , Pregnanolone/pharmacology , Pyridazines/metabolism , Pyridazines/pharmacology , Receptors, GABA-A/metabolism , Sulfur Radioisotopes , Tritium , Zinc/metabolism , Zinc/pharmacology , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacology
17.
Mol Pharmacol ; 51(4): 588-96, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9106623

ABSTRACT

The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.


Subject(s)
Anesthetics, Inhalation/pharmacology , Ethanol/pharmacology , GABA Modulators/pharmacology , Gene Deletion , Pentobarbital/pharmacology , Receptors, GABA-A/physiology , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Cerebellum/cytology , Cerebellum/ultrastructure , Enflurane/pharmacology , Female , Halothane/pharmacology , Macromolecular Substances , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Polymerase Chain Reaction , Rats , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Transcription, Genetic
18.
Proc Natl Acad Sci U S A ; 94(8): 4143-8, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9108119

ABSTRACT

gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The beta3 subunit is an essential component of the GABA(A)-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a beta3 gene inactivated by gene targeting. GABA(A)-R density is approximately halved in brain of beta3-deficient mice, and GABA(A)-R function is severely impaired. Most beta3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. beta3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the beta3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. beta3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.


Subject(s)
Angelman Syndrome/genetics , Cleft Palate/genetics , Epilepsy/genetics , Receptors, GABA/physiology , Animals , Behavior, Animal/physiology , Cleft Palate/physiopathology , Epilepsy/physiopathology , Gene Targeting , Humans , Mice , Mice, Knockout
19.
Anesthesiology ; 86(3): 538-48, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9066319

ABSTRACT

BACKGROUND: Although previous studies have provided some insight into the pharmacologic aspects of nitrous oxide analgesia, the neural circuits mediating its antinociceptive effect remain relatively unexplored. Position emission tomography was used in nine volunteers to identify the loci of nitrous oxide-modulated cerebral responses to a peripheral noxious stimulus. METHODS: Nitrous oxide-pain interactions were studied by comparing regional cerebral blood flow responses to a 48 degrees C tonic heat stimulus, applied to each volunteer's left forearm, during room air inhalation with those obtained while 20% nitrous oxide was administered. Two cerebral blood flow scans were obtained with the 15O-water technique during each condition. Locations of specific regional activation related to pain, and nitrous oxide, were identified using the statistical parametric mapping method, with a significance level of P < 0.01. Pain was rated by visual analog scale and the values were compared using Wilcoxon rank sum analysis. RESULTS: Pain produced cerebral activation in the contralateral thalamus, anterior cingulate, and supplementary motor area. Adding nitrous oxide during pain stimulation abolished activation in these areas but was associated with activation in the contralateral infralimbic and orbitofrontal cortices. In parallel, mean visual analog scale scores decreased significantly from 67 +/- 4 (SEM) to 54 +/- 5 (P < 0.05). CONCLUSIONS: Nitrous oxide, at 20% concentration, appears to modulate pain processing in the brain's medial pain system, and also activates the infralimbic and orbitofrontal cortices. The potential contribution of the affected brain areas to nitrous oxide analgesia is discussed.


Subject(s)
Analgesics, Non-Narcotic/pharmacology , Brain/drug effects , Brain/diagnostic imaging , Nitrous Oxide/pharmacology , Administration, Inhalation , Adult , Analgesics, Opioid/pharmacology , Brain/physiology , Cerebrovascular Circulation/drug effects , Dose-Response Relationship, Drug , Female , Glucose/metabolism , Heating , Humans , Male , Nociceptors/drug effects , Nociceptors/physiology , Oxygen Radioisotopes , Pain/drug therapy , Pain/physiopathology , Synapses/drug effects , Synapses/physiology , Tomography, Emission-Computed
20.
Anesth Analg ; 84(1): 120-6, 1997 Jan.
Article in English | MEDLINE | ID: mdl-8989012

ABSTRACT

Recent positron emission tomography (PET) studies have demonstrated areas of pain processing in the human brain. Given the inhibitory effects of opioids on neuronal activity, we predicted that fentanyl's analgesic effects would be associated with suppression of pain-evoked responses in these distinct brain areas. To test this, PET was used to measure cerebral blood flow responses, as reflections of regional neuronal activity, to painful and nonpainful thermal stimuli both in the absence and presence of fentanyl in humans. During each PET scan in nine healthy volunteers a tonic heat source was placed against the subject's left forearm, delivering a preset temperature of either 40 degrees C (nonpainful) or 47-48 degrees C (painful). Subjects underwent eight blood flow studies, each consisting of 50 mCi [15O]water injection and a PET scan. The first four studies were performed during placebo administration in the stimulus sequence: nonpainful, painful, painful, nonpainful. This sequence was then repeated during intravenous (i.v.) administration of fentanyl 1.5 micrograms/kg [corrected]. Significant differences in regional cerebral blood flow (rCBF) between the placebo and the fentanyl conditions during nonpainful and painful stimuli were identified using statistical parametric mapping. It was found that pain increased rCBF in the anterior cingulate, ipsilateral thalamus, prefrontal cortex, and contralateral supplementary motor area. Fentanyl increased rCBF in the anterior cingulate and contralateral motor cortices, and decreased rCBF in the thalamus (bilaterally) and posterior cingulate during both stimuli. During combined pain stimulation and fentanyl administration, fentanyl significantly augmented pain-related rCBF increases in the supplementary motor area and prefrontal cortex. This activation pattern was associated with decreased pain perception, as measured on a visual analog scale. In contrast to our hypothesis, these data indicate that fentanyl analgesia involves augmentation of pain-evoked cerebral responses in certain areas, as well as both activation and inhibition in other brain regions unresponsive to pain stimulation alone.


Subject(s)
Analgesics, Opioid/pharmacology , Brain/diagnostic imaging , Fentanyl/pharmacology , Tomography, Emission-Computed , Adult , Brain/drug effects , Brain/physiopathology , Cerebrovascular Circulation/drug effects , Female , Fentanyl/administration & dosage , Hot Temperature , Humans , Injections, Intravenous , Male , Pain/physiopathology , Pain Measurement , Perception
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